POS0616 THE CHARACTERISTICS OF PROGRESSIVE FIBROSING INTERSTITIAL LUNG DISEASE IN CONNECTIVE TISSUE DISEASES WITH SINGLE-CELL RNA SEQUENCING OF BRONCHOALVEOLAR LAVAGE FLUID
Authors: W. Fujii, A. Sakashita, A. Hirano, K. Baßler, M. Kadoya, A. Omoto, W. Fukuda, T. Seno, M. Wada, M. Kohno, Y. Kawahito
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Connective tissue disease-associated interstitial lung disease (CTD-ILD) is a severe complication of rheumatic and musculoskeletal diseases, e.g. rheumatoid arthritis (RA), dermatomyositis (DM), systemic sclerosis (SSc) and so on. CTD-ILD is a heterogeneous syndrome driven by different diseases and cell types. This complexity provides a challenging field for elucidating the mechanism of the disease. Although some patients with CTD-ILD develop progressive fibrosing interstitial lung disease (PF-ILD) like as other ILD despite immunosuppressive treatment, it is difficult to predict which patients develop PF-ILD before treatment. <h3>Objectives</h3> The objective of this study is to find characteristics of immune cells and predictive biomarkers of PF-ILD in CTD-ILD patients. <h3>Methods</h3> We collected BALF and blood from 30 CTD-ILD and 12 idiopathic interstitial pneumonia (IIP) patients before treatment. Twelve out of 42 patients fulfilled PF-ILD criteria<sup>[1]</sup> in 1 year period. PF-ILD patients included 3 RA, 2 DM, 2 Sjögren syndrome, 1 ANCA-associated vasculitis, and 4 IIP patients. We applied Seq-Well, a portable platform of single-cell RNA sequencing<sup>[2]</sup>, to analyze gene expressions in immune cells in BALF and blood. We compared the distribution of immune cells and differential gene expressions in BALF and blood between PF-ILD patients and patients without PF-ILD in CTD-ILD and IIP patients. <h3>Results</h3> We found that alveolar macrophages are the most abundant cell types in BALF of PF-ILD patients. We further classified alveolar macrophages and neutrophils into more detailed subsets based on their gene expression patterns and compared them between PF-ILD and patients without PF-ILD. In PF-ILD patients, we found increased CXCL10<sup>+</sup>, CXCL8<sup>+</sup> alveolar macrophages and complement activation associated genes were upregulated in BALF. Moreover, we found increased interferon-induced proteins with tetratricopeptide repeats (IFIT)<sup>+</sup>, MMP-9<sup>+</sup> and immature neutrophils, which were reported to be associated with severe COVID-19 pneumonia<sup>[3]</sup>, in the blood of PF-ILD patients. Differentially expressed gene analysis revealed high levels of high mobility group box (HMGB)-2, IL-1β, and complement activation associated genes in alveolar macrophages in PF-ILD patients. <h3>Conclusion</h3> Alveolar macrophage phenotypes were changed in BALF while neutrophil phenotypes were changed in the blood of PF-ILD in CTD-ILD and IIP patients, which are consistent with the characteristics of severe COVID-19 pneumonia patients, suggesting common pathological pathways of lung fibrosis. We propose the hypothesis that alveolar macrophage phenotypic changes lead to complement and cytokine/chemokine mediated neutrophil activation and progression of PF-ILD (Figure 1). These cell subpopulations and up-regulated genes would be potential biomarkers or therapeutic target of PF-ILD in CTD-ILD patients. <h3>References</h3> [1]Flaherty KR et al, Nintedanib in Progressive fibrosing interstitial lung diseases. New Engl J Med 2019; 381(18):1718-1727. [2]Gierahn TM et al, Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput. Nat Methods 2017;14(4):395-398. [3]Schulte-Schrepping J et al, Severe COVID-19 is marked by a dysregulated myeloid cell compartment. Cell 2020; 182(6): 1419-1440. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> Wataru Fujii Speakers bureau: Boehringer Ingelheim Japan, Inc., Mitsubishi Tanabe Pharma Corporation, Grant/research support from: Boehringer Ingelheim Japan, Inc., GlaxoSmithKline K.K., Takeda Pharmaceutical Co.,Ltd., Aki Sakashita: None declared, Aiko Hirano Speakers bureau: Sanofi K.K., Kevin Baßler: None declared, Masatoshi Kadoya: None declared, Atsushi Omoto Speakers bureau: AbbVie GK, Chugai Pharmaceutical Co. Ltd., Ono Pharmaceutical Co., Ltd., Daiichi-Sankyo Inc., Astellas Pharma Inc., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Asahi Kasei Pharma Corp, Novartis Pharma KK, Gilead Sciences Inc., Janssen Pharmaceutical K.K., GlaxoSmithKline K.K, Eli Lilly Japan K.K, Wataru Fukuda Speakers bureau: Chugai Pharmaceutical Co., Eisai Co., Ltd., Eli Lilly Japan K.K., AbbVie GK, Ono Pharmaceutical Co. Ltd., Astellas Pharma Inc., UCB Japan Co. Ltd., Pfizer Japan Inc., Giread Science K.K., Asahikasei Pharma Co., Daiichi-Sankyo Inc., Takahiro Seno Speakers bureau: Asahi Kasei Pharma, Janssen Pharmaceutical K.K., Boehringer Ingelheim Japan, Inc, Makoto Wada Speakers bureau: AbbVie GK, Astellas Pharma Inc., Bristol Myers Squibb Co., Chugai Pharmaceutical Co. Ltd., Eisai Co. Ltd., Eli Lilly Japan K.K, Janssen Pharmaceutical K.K., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Sanofi Japan K.K., Masataka Kohno Speakers bureau: AbbVie GK, Astellas Pharma Inc., Bristol Myers Squibb Co., Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co., Eli Lilly Japan K.K, Janssen Pharmaceutical K.K., Mitsubishi Tanabe Pharma Co., Asahikasei Pharma Co., Pfizer Japan Inc., Sanofi Japan K.K., Boehringer Ingelheim Co. Ltd., UCB Japan Co. Ltd., Eisai Co. Ltd., GlaxoSmithKline K.K., Yutaka Kawahito Speakers bureau: AbbVie GK, Asahi Kasei Pharma Corp, Astellas Pharma Inc., Ayumi Pharmaceutical Corp., Boehringer Ingelheim Japan Inc., Bristol Myers Squibb Co., Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co., Eisai Co. Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Takeda Pharmaceutical Co. Ltd., Teijin Pharma Ltd., UCB Japan Co. Ltd., GlaxoSmithKline K.K., Eli Lilly Japan K.K, Janssen Pharmaceutical K.K., Sanofi Japan K.K., Gilead Sciences Inc., Mylan EPD, Grant/research support from: AbbVie GK, Asahi Kasei Pharma Corp, Astellas Pharma Inc., Ayumi Pharmaceutical Corp., Boehringer Ingelheim Japan Inc., Bristol Myers Squibb Co., Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co., Eisai Co. Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Takeda Pharmaceutical Co. Ltd., Teijin Pharma Ltd., GlaxoSmithKline K.K., Gilead Sciences Inc.